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. 2012 Sep 12;7(9):e44690. doi: 10.1371/journal.pone.0044690

Figure 3. Direct chromatin PCR for CD34 gene: detection of epigenetic changes during cellular differentiation in vitro.

Figure 3

Primer locations for DC-PCR are shown on a map of the CD34 5′ region (A). An arrow indicates the transcription start site for transcript NM_001025109.1. Flow cytometry (Left panel) and DC-PCR analysis (Right panel) of cord blood CD34+ cells as they undergo cytokine mediated differentiation in vitro (B). One day after CD34 magnetic bead purification about 65% of cells expressed CD34 as determined by flow cytometry (Left panel). At day 15 this fraction had decreased to 37.1%, while cells remained healthy with less than 10% showing evidence for loss of membrane integrity as assessed by 7-AAD co-staining (Left panel). CD34 DC-PCR (Right panel) identified decreased accessibility of sequences within about 500 bp 5′ of the CD34 transcription start site on day 15 compared to day 1 and of 11 tested sequences in this region (primers 9–19) only 3 yielded (faint) DC-PCR products in CD34 negative (Left panel) KMS-12-PE cells (Right panel). Results are representative of three independent experiments.